IMPROVED ASSAY FOR PLASMA DIHYDROXYPHENYLACETIC ACID AND OTHER CATECHOLS USING HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY WITH ELECTROCHEMICAL DETECTION

Several modifications of an HPLC-electrochemical assay method for plas ma levels of norepinephrine (NE), epinephrine (EPI), dopamine (DA), di hydroxyphenylglycol (DHPG), dihydroxyphenylalanine (DOPA) and dihydrox yphenylacetic acid (DOPAC) that improve the accuracy and reliability o f DHPG, DOPA, and DOPAC measurements are described. In batch alumina e xtractions, increasing the amount of alumina decreased analytical reco veries of DHPG, DOPA, and especially DOPAC, and increasing the strengt h of the eluting acid increased recoveries of these catechols, without affecting recoveries of the amines NE, EPI and DA. Refrigeration (4-d egrees-C) until injection stabilized DOPAC in aqueous solution and the refore improved the reproducibility of plasma DOPAC measurements. Circ ulation of chilled water (15-degrees-C) around the column using a wate r jacket decreased variability in retention times of the catechols and thereby facilitated identification of peaks, while enhancing separati on of DHPG from the solvent front. Use of 6-fluoro-DOPA and 6-fluoro-D OPAC as internal standards did not improve inter-assay reliability. We recommend that in assays of plasma catechols including DOPAC, small ( 5 mg), precisely measured amounts of alumina be used, with a relativel y strong eluting solution (e.g. 0.04 M phosphoric acid-0.2 M acetic ac id, 20:80, v/v), and that the samples be refrigerated until injection, with column temperature held constant at less than 20-degrees-C.